Rational Reprogramming of O-Methylation Regioselectivity for Combinatorial Biosynthetic Tailoring of Benzenediol Lactone Scaffolds

Article abstract of DOI:10.1021/jacs.8b12967

O-Methylation modulates the pharmacokinetic and pharmacodynamic (PK/PD) properties of small-molecule natural products, affecting their bioavailability, stability, and binding to targets. Diversity-oriented combinatorial biosynthesis of new chemical entiti

Full text of DOI:10.1021/jacs.8b12967

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Article
Rational reprogramming O-methylation regioselectivity for
combinatorial biosynthetic tailoring of benzenediol lactone scaffolds
Xiaojing Wang, Chen Wang, Lixin Duan, Liwen Zhang, Hang Liu, Ya-Ming Xu, Qingpei Liu, Tonglin
Mao, Wei Zhang, Ming Chen, Min Lin, A. A. Leslie Gunatilaka, Yuquan Xu, and Istvan Molnar
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b12967 • Publication Date (Web): 15 Feb 2019
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Rational reprogramming O-methylation regioselectivity for combinatorial biosynthetic
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tailoring of benzenediol lactone scaffolds
Xiaojing Wanga,b,c,1, Chen Wanga,b,1, Lixin Duanb,d,1, Liwen Zhanga, Hang Liua,b, Ya-ming
Xub, Qingpei Liub,e, Tonglin Maoc, Wei Zhanga, Ming Chena, Min Lina, A.A. Leslie Gunatilakab,
Yuquan Xua*, and István Molnárb*
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a Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, 12 Zhongguancun
South Street, Beijing 100081, P.R. China
b
Southwest Center for Natural Products Research, University of Arizona, 250 E. Valencia Rd.,
Tucson, AZ 85706, USA
c
State Key Laboratory of Plant Physiology and Biochemistry, Department of Plant Sciences,
College of Biological Sciences, China Agricultural University, Beijing 100193, P.R. China
d
Guangzhou University of Chinese Medicine, 232 Waihuan East Road, Guangzhou University
City, Panyu District, Guangzhou 510006, P.R. China
e Key Laboratory of Environment Correlative Dietology, College of Food Science and Technology
, Huazhong Agricultural University , Wuhan 430070 , P.R. China
1 These authors contribute equally to this work.
* Corresponding authors: I. Molnár, Tel: +1 (520) 621-9932, Email: imolnar@email.arizona.edu;
and Y. Xu, Tel: +86 (010) 8210-9850, Email: xuyuquan@caas.cn
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Abstract
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O-Methylation modulates the pharmacokinetic and pharmacodynamic (PK/PD) properties of
small molecule natural products affecting their bioavailability, stability, and binding to targets.
Diversity-oriented combinatorial biosynthesis of new chemical entities for drug discovery, and
optimization of known bioactive scaffolds during drug development both demand efficient O-
methyltransferase (OMT) biocatalysts with considerable substrate promiscuity and tunable
regioselectivity that can be deployed in a scalable and sustainable manner. Here we demonstrate
efficient total biosynthetic and biocatalytic platforms that use a pair of fungal OMTs with
orthogonal regiospecificity to produce unnatural O-methylated benzenediol lactone (BDL)
polyketides. We show that rational, structure-guided active site cavity engineering can reprogram
the regioselectivity of these enzymes. We also characterize the interplay of engineered
regioselectivity with substrate plasticity. These findings will guide combinatorial biosynthetic
tailoring of unnatural products towards the generation of diverse chemical matter for drug discovery
and the PK/PD optimization of bioactive scaffolds for drug development.
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Introduction
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Alkylations, most importantly the methylation of O-, C-, N- and S-centered nucleophiles are
ubiquitous tailoring reactions during the biosynthesis of all major classes of small molecule natural
products.1 Among these reactions, O-methylation is used by nature and medicinal chemistry for
the hydrophobic masking of peripheral hydroxyl or carboxylic acid moieties to adjust the
physicochemical and biological properties of bioactive compounds. The resulting “methyl effect”
increases the lipophilicity and membrane permeability of small molecule scaffolds, enhancing their
membrane transport, oral bioavailability, absorption and excretion.2,3 It also modulates the
reactivity of small molecules by umpolung (reversal of polarity) of hydroxyl groups,4 affecting the
in vivo metabolic stability of drugs and channeling the biosynthesis of natural products through a
series of reactive intermediates.5,6 O-Methylation may also alter the conformation of natural
product scaffolds through stereoelectronic and steric effects and confers weak interactions that
contribute to binding to various targets including receptors.7 As opposed to chemical synthesis,
enzymatic O-methylation offers regio- and stereospecific outcomes even for highly complex and
reactive scaffolds without resorting to expensive multistep protection/deprotection strategies. It
also proceeds with a high yield under mild, environmentally friendly reaction conditions, and does
not have to contend with the formation of C-methylated side products in the case of phenolic
substrates.8,9 In the biotechnology industries, O-methylation is conducted by utilizing in vivo
biocatalytic platforms instead of in vitro reconstituted enzymatic reactions because the availability
of the methyl donor co-substrate S-adenosyl-methionine (SAM) is limited and its in vitro
regeneration is inefficient.1,10
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O-Methylation has been engineered in bacterial natural product biosynthetic pathways,7
mostly those of polyketides and nonribosomal peptides, by deleting the genes encoding native O-
methyltransferase (OMT) enzymes or augmenting the pathways with genes for heterologous OMTs
despite the often-constrained substrate flexibility of these enzymes. Extensive work has also been
conducted on dissecting and altering the chemo- and regioselectivities of the mammalian and
bacterial catechol OMTs, and those of the plant and bacterial flavonoid and phenylpropanoid
OMTs.7,10 Such studies show that regio- and chemospecificity are intimately connected in each
enzyme scaffold,11–13 although divorcing these properties is possible to some extent by directed
evolution or active site cavity engineering informed by enzyme-substrate docking studies.14–17
However, little attention has been paid up to now to define the substrate promiscuity and modulate
the regioselectivity of fungal secondary metabolite OMTs, despite these enzymes constituting the
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most common tailoring activities during the biosynthesis of such natural products.
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OMTs are also crucial to modulate the biological activities of benzenediol lactones (BDLs), a
growing group of fungal polyketide natural products with an astonishing range of biological
activities.18,19 BDLs are defined by a 1,3-benzenediol moiety fused to a macrocyclic lactone ring,
with C2–C7 connectivity as in resorcylic acid lactones (RALs) or with C3–C8 connectivity as in
dihydroxyphenylacetic acid lactones (DALs). Natural RALs contain 12- or 14-membered
macrocycles (RAL12 or RAL14), while natural DALs most often include 12-membered
macrolactones (DAL12). Amongst RAL12, lasiodiplodin (1a) with a 3-methoxy moiety displays
ATP- and prostaglandin synthesis inhibitory activities, while its unmethylated congener, 3-
desmethyl-lasiodiplodin (DLD, 1) is devoid of such activities. Instead, DLD 1 is a nonsteroidal
mineralocorticoid receptor antagonist and pancreatic lipase inhibitor.20 RAL14 with a 5-methoxy
group are also important pharmacophores. Thus, hypothemycin is a selective inhibitor of mitogen-
associated protein kinases; 7-oxozeaenol inhibits the tumor growth factor β activated kinase 1;
while neocosmosin C is an agonist of δ-opioid receptors.18,19 Conversely, BDLs devoid of O-
methylation also reveal exciting activities. Thus, RAL14 in the pochonin/monocillin/radicicol
family are potent Hsp90 inhibitors; zearalenone is an estrogen agonist; and the DAL12 curvularins
display anti-inflammatory and induced nitric oxide synthase inhibitory activities.18,19
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The polyketide scaffolds of these and other BDLs are assembled in fungi by two iterative Type
I polyketide synthases (iPKSs) that collaborate as BDL synthase (BDLS) subunits, and are further
tailored by oxidations, reductions, halogenations, and O-methylations to afford the mature
bioactive molecules.21–23 We have been developing combinatorial biosynthetic platforms that
exploit the flexibility of secondary metabolic biosynthetic enzymes to generate structurally
complex molecules that may be utilized for drug discovery and agricultural applications. We have
generated novel BDL scaffolds in the heterologous host Saccharomyces cerevisiae by BDLS
subunit shuffling, combinatorial domain exchanges and rational engineering of the active sites of
domains,24–27 and elaborated these biosynthons by exploiting combinatorial tailoring reactions in
total biosynthetic or biocatalytic platforms.28 Here, we evaluate the combinatorial biosynthetic
potential of two orthologous OMT enzymes that afford orthogonal regioisomeric outcomes when
modifying the same BDL substrate. Using protein homology modeling and substrate docking, we
show that subtle differences in the active site cavity architecture of these OMTs force the substrates
to approach the conserved active site base in distinct poses. Rational engineering of these
“plasticity residues” then allows us to morph the regioselectivity of one enzyme to that of the other,
and to create nonselective or iterative OMT variants. These experiments also reveal that
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regioselectivity is influenced by the substrate, thus engineering artificial BDL biosynthetic
pathways demands OMTs adapted for the chosen scaffold and optimized to yield the desired
regioisomer.
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Results and Discussion
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Regiospecificity and substrate selectivity of LtOMT and HsOMT during
combinatorial biosynthesis.
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Previous studies identified LtOMT and HsOMT, two orthologous O-methyltransferases
(OMTs) encoded in the biosynthetic gene clusters for two fungal benzenediol lactones (BDLs).21,29
LtOMT from Lasiodiplodia theobromae methylates the C-3 phenolic hydroxyl of the RAL12
desmethyl-lasiodiplodin (DLD, 1) to produce lasiodiplodin (1a, Fig. 1).21 HsOMT (Hpm5) from
Hypomyces subiculosus methylates the C-5 phenolic hydroxyl of the RAL14 trans-14,15-
dehydrozearalenol (DHZ, 3) to afford 5-O-methyl DHZ 3b (Fig. 1), an intermediate of
hypothemycin.29 To investigate whether these apparently different regiospecificities are intrinsic
to the enzymes or dictated by the substrates, we used a Saccharomyces cerevisiae host to co-express
LtOMT in a heterocombination with the DHZ-synthesizing iPKS pair, and separately, HsOMT
with the DLD-producing iPKSs. Gratifyingly, both OMTs accepted the BDLs offered by the
noncognate iPKS pairs and efficiently processed each of these compounds to a single O-methylated
product. Thus, LtOMT afforded 3-O-methyl-DHZ 3a, while HsOMT yielded 5-O-methyl-DLD 1b:
these derivatives are the regioisomers of the cognate OMT products 5-O-methyl DHZ 3b and
lasiodiplodin 1a, respectively (Fig. 1). This suggested that the size, shape and functionalization of
the macrocyclic ring may not affect the regiospecificities of these enzymes. Co-expression of both
OMTs in a single chassis with the DLD- or the DHZ-producing iPKS pairs yielded mixtures of the
3-O-methylated, the 5-O-methylated and the 3,5-O-dimethylated products 1a, 1b, and 1c, or 3a,
3b, and 3c, respectively. This indicated that at least one of the OMTs is able to accept the
methylated product of the other OMT as an alternative substrate.
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Figure 1. Total biosynthesis of O-methylated BDLs using LtOMT and HsOMT. Product
profiles (reversed-phase LC-MS traces recorded at 210 nm with a photodiode array detector) are
shown for cultures of S. cerevisiae BJ 5464-NpgA strains co-transformed with the relevant iPKS
pairs,21,29 and with LtOMT and/or HsOMT as indicated. The identities of the products were
validated by comparison with isolated and structurally characterized compounds.
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To further explore the breadth of substrate tolerance and the fidelity of regioselectivity of these
two OMTs, we assembled a collection of model substrates that represent the natural and “unnatural”
(combinatorial biosynthetic) structural space of BDLs (Fig. 2 and SI Appendix, Fig. S1), including
RAL12 (DLD 1, radiplodin 2 and trans-resorcylide), RAL14 (DHZ 3, zearalenol 4, lasicicol 5,
radicicol, monocillin I and monocillin II), DAL12 (10(11)-dehydrocurvularin and epi-
dehydrocurvularin) and DAL14 (lasilarin 6 and radilarin). To explore the requirement for a
macrolactone ring, we also tested non-macrocyclic BDL congeners such as isocoumarins (IC15,
IC18 and IC28), acyl resorcylic acids (ARA7 7 and ARA8 8), an acyl dihydroxybenzoic acid
(ADA5) and a benzaldehyde (pre-asperfuranone). Initial testing of these potential substrates was
conducted in a total biosynthetic format by co-expressing the appropriate iPKS pairs with the
OMTs. Successful reactions were repeated using a standardized biocatalytic platform whereby the
overall rate of O-methylation and the product distribution was quantified after feeding purified
substrates to the OMT-expressing yeast host.
The two OMTs displayed considerable substrate flexibility. Thus, six out of the 20 BDL
congeners were successfully O-methylated by both LtOMT and HsOMT, with an additional two
compounds also processed by LtOMT (Fig. 2 and SI Appendix, Fig. S1). Remarkably, differences
in the geometry of the benzenediol lactone ring system (RAL vs. DAL), variations in the size (12-
or 14-membered) or the functionalization of the macrocycle, or even the absence of the macrocyclic
ring were all well tolerated. However, α-pyrone and benzaldehyde analogues were not accepted,
nor could the substrate be simplified to a single phenolic ring as shown by the absence of O-
methylation of orsellinic acid, 2,4-dihydroxybenzoic acid, resorcinol or phenol (SI Appendix, Fig.
S1). For macrocyclic substrates, the presence of an enone moiety at C9-C11 was found to be
incompatible with O-methylation by LtOMT or HsOMT, as shown by radicicol, monocillin I,
monocillin II, trans-resorcylide, radilarin, 10(11)-dehydrocurvularin and epi-dehydrocurvularin (SI
Appendix, Fig. S1).
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Figure 2. Combinatorial tailoring with LtOMT and HsOMT. Stacked polar bar charts
show the product distributions among o-methoxy (blue), p-methoxy (green) and o,p-dimethoxy
(red) BDL congeners during biocatalytic transformation of the indicated substrates (bold numbers
above the bars) with S. cerevisiase BJ5464-NpgA expressing LtOMT (substrates 1-8, 1b-4b and
6b-8b); HsOMT (substrates 1-8 and 1a-8a); and both LtOMT and HsOMT (substrates 1-8).
Percent values below the compound numbers on these charts show the overall conversion of the
indicated substrate into all corresponding methylated products. See SI Appendix, Table S3 for
tabulated percent conversion values as the mean ± SD from three independent experiments of three
replicates each (n=9). The structures of substrates 1-8 are shown with the carbonyl “handle” (see
text for details) highlighted in yellow, and the sites of O-methylation identified by blue (LtOMT-
type ortho-OMT activity) and green ovals (HsOMT-type para-OMT activity). Scheme:
Unmethylated substrates (X) may be converted to the o-methoxy congener (Xa) by the LtOMT,
and to the p-methoxy congener (Xb) by the HsOMT enzyme. Xa and Xb may also be converted
to the o,p-dimethoxy congener (Xc) by the appropriate enzyme (X=1-8). See SI Appendix Fig. S2
for examples of the resulting biocatalytic grids.
HsOMT is apparently more fastidious than LtOMT in accepting noncognate substrates. First,
neither radiplodin (RDN, 2) nor lasicicol (LCL, 5) is O-methylated by this enzyme (Fig. 2). For
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RDN 2, the highly constrained macrocycle may be sterically incompatible with the active site
pocket of the enzyme. For LCL 5, enolization of the C-9 carbonyl allows the resulting hydroxyl
group to serve as a proton donor, and the resulting spurious H-bond(s) may restrict the substrate
from assuming a productive orientation within the active site cavity of the enzyme. Although a
carbonyl is also present equidistant to the target hydroxyl group in the acceptable substrate lasilarin
(LLN 6, the skeletal isomer of LCL 5), that carbonyl at C-1 is part of the lactone and thus would
not be subject to enolization. Even for those noncognate substrates that were accepted by HsOMT,
the conversion rates were frequently inferior to those of LtOMT with the same scaffold, as seen
with DLD 1, zearalenone (ZEA, 4), and to a lesser extent with ARA7 7 and LLN 6 (Fig. 2, SI
Appendix, Table S3).
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The 14 resulting O-methylated products, including 11 that are new to nature were isolated,
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and their structures were elucidated by comparison of their H and 13C NMR data with those
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reported, and by careful analysis of their 1D NOESY, H-1H COSY, HSQC, and HMBC NMR
spectra to ascertain the position of the methoxy moieties (SI Appendix, Fig. S6 and Table S2). For
all accepted substrates, O-methylation regioselectivity was found to be idiosyncratic for the two
OMTs, despite the large structural differences of the BDL congeners (Fig. 2). Thus, LtOMT is
specific for the phenolic hydroxyl residing at the ortho position to the aromatic carbon bearing the
carbonyl (hereafter, ortho-OMT activity). Conversely, HsOMT O-methylates the phenolic
hydroxyl that is at the para position to the same (henceforth, para-OMT activity). The carbonyl
“handle” for this recognition can be part of a lactone, an ester or a ketone. No “off-target” O-
methylation was observed with either enzyme. This strict regiospecificity is in contrast to the
chemical (non-enzymatic) O-methylation of phenolic hydroxyl groups which either affords a
mixture of methoxy regioisomers and di-substituted products, or requires laborious protection-
deprotection reactions to achieve regioselectivity.30
Mono-O-methylated congeners as substrates for subsequent combinatorial O-
methylation.
We also investigated whether preexisting O-methylation at one phenolic hydroxyl group
would influence the suitability of a scaffold towards subsequent O-methylation at the other
hydroxyl (Fig. 2, SI Appendix, Table S3). To do this, we compared results from three biocatalytic
systems. First, we offered o-methoxy BDL congeners to the S. cerevisiae strain expressing HsOMT.
Second, we provided p-methoxy compounds to the yeast host producing LtOMT. Third, we
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interrogated O-methylation of unmethylated substrates by yeast strains co-expressing LtOMT and
HsOMT in the same chassis. These experiments revealed that seemingly very similar final product
distributions can result from very different processes in the coupled biocatalytic production system
where two enzymes are co-expressed in the same host chassis and compete for the same
unmethylated substrate, and where the mono-O-methylated product of either enzyme may or may
not serve as a second-stage substrate for the other enzyme (Fig. 2). The resulting six o,p-dimethoxy
BDL congeners, all of which are new to nature were isolated and their structures were elucidated
by 1D and 2D NMR spectroscopy (SI Appendix, Table S2 and Fig. S6).
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In some cases, preexisting O-methylation severely reduced the ability of both OMTs to accept
a BDL scaffold as a substrate, as was seen with O-methylated LLN 6 congeners (Fig. 2, SI Appendix,
Fig. S2A and Table S3). Consequently, only a small amount of the dimethoxy LLN analog 6c was
made in the LtOMT-HsOMT co-expression system. In other cases, preexisting O-methylation had
no effect on the ability of either LtOMT or HsOMT to carry out subsequent methylation of the
other phenolic hydroxyl, as was observed with methoxy congeners of the DLD 1 scaffold (Fig. 2,
SI Appendix, Fig. S2B and Table S3). Thus, the dimethoxy DLD congener 1c could be made either
through the 11a1c or the 11b1c route. However, since O-methylation of 1/1a by HsOMT
is far less complete than that of 1/1b by LtOMT, lasiodiplodin 1a continued to accumulate in the
LtOMT-HsOMT co-expression system, while 5-O-methyl DLD 1b was mostly converted to the
dimethoxy DLD congener 1c.
Preexisting O-methylation was often inhibitory to HsOMT but not to LtOMT (Fig. 2, SI
Appendix, Fig. S2C and Table S3). Thus, ortho-O-methylation (as in 3-O-methyl DHZ 3a, 3-O-
methyl ZEA 4a, 3-O-methyl ARA7 7a, and 3-O-methyl ARA8 8a) prevented further O-
methylation by HsOMT, while the regioisomeric congeners (such as 5-O-methyl DHZ 3b, 5-O-
methyl ZEA 4b, 5-O-methyl ARA7 7b, and 5-O-methyl ARA8 8b) remained excellent substrates
for LtOMT. As a result, the corresponding o,p-dimethoxy derivatives could only be made via the
XXbXc route in the LtOMT-HsOMT co-expression system (X=3, 4, 7, or 8). At the same
time, most of the available substrates remained trapped as the ortho-O-methyl congeners (as with
3-O-methyl ZEA 4a, 3-O-methyl ARA7 7a, and 3-O-methyl ARA8 8a) since these could not be
further converted by HsOMT. A contrary situation was observed for RDN 2 that is not a substrate
for HsOMT (Fig. 2, SI Appendix, Fig. S2D and Table S3). Here, pre-existing ortho-O-methylation
(as in 3-O-methyl RDN 2a) made this scaffold acceptable, albeit only marginally, to serve as a
substrate for HsOMT. Thus, a small amount of the o,p-dimethoxy RDN derivative 2c was obtained
through the 22a2c route in the co-expression system. Since 5-O-methyl RDN 2b, an otherwise
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excellent substrate for LtOMT, was not produced by HsOMT, the dimethoxy RDN congener 2c
could not be made by the 22b2c route.
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Docking identifies distinct substrate binding poses.
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Despite the mutually orthogonal regiospecificities of LtOMT and HsOMT against multiple
and structurally varied substrates, these enzymes are orthologous with a relatively high sequence
similarity (58% identity). This suggests that the target site for O-methylation may be determined
by a limited number of key amino acid residues. To identify the structural basis for the
programming of these distinct regiospecificities, we constructed homology protein structure
models for LtOMT and HsOMT (Fig. 3) based on the known crystal structure of the mitomycin 7-
O-methyltransferase of Streptomyces lavendulae (MmcR, PDB 3GXO;31 Z-score 43.3, rmsd 0.9,
and identity of 26% with LtOMT; and Z-score 41.6, rmsd 2.1, and identity of 24% with HsOMT).
Both OMT models feature an N-terminal dimerization domain each (amino acids 1-130, SI
Appendix, Fig. S3), and a C-terminal OMT Class I, α/β Rossmann-fold catalytic domain with the
conserved SAM-binding signature motif DxGGGxG (amino acids 231-237)31–33. Modeling of S-
adenosyl-L-homocysteine (SAH) into LtOMT and HsOMT indicated that the key enzyme-
coproduct interactions are well conserved amongst LtOMT, HsOMT, MmcR31 and similar MTs
such as the calicheamicin orsellinic acid methyltransferase CalO1 of Micromonospora
echinospora32 (SI Appendix, Fig. S4). Thus, the adenine moiety of SAH is predicted to be stabilized
by π-stacking with F284, while the sulfur atom of SAH could engage in hydrophobic interactions
with F197 and M201. The SAH adenine N-6 amino group forms a hydrogen bond with D283,
while the ribose O-2 and O-3 may engage in a hydrogen bonding network with R262 and E261,
with the latter residue also interacting with the SAH adenine N-3. Finally, the terminal carboxylic
acid group of SAH may form hydrogen bonds with E203 while the terminal amino group could
interact with the main chain carbonyl oxygen of G233.
Docking of DLD 1 and DHZ 3 indicated that, similar to other phenolic OMT enzymes31,32
,
substrate binding is dominated by hydrophobic and van der Waals interactions with residues such
as F52* and W55* from the other subunit of the dimer (for both enzymes); residues F197, M201,
A200 and M344 (for LtOMT); and amino acids V136, F197, M201 and I349 (for HsOMT).
Importantly, docking also revealed characteristically different binding poses for these substrates in
LtOMT vs. HsOMT (Fig. 3, SI Appendix Fig. S4). For LtOMT, both DLD and DHZ are predicted
to present their OH-3 to the Nε of the deduced active site base H30333, while OH-5 may be
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contacted by the main-chain carboxyl group of D202. Conversely, rotation of both substrates
within the active site cavity may position OH-5 for attack by H303 of HsOMT, while OH-3 and
the C-1 carbonyl could interact with the side-chain hydroxyl group of S200. Abstraction of the
proton from the substrates by H303 of either enzyme would then lead to a nucleophilic attack of
the phenolate anion of the substrate on the reactive methyl group of the co-substrate S-adenosyl-L-
methionine (SAM). Meanwhile, the catalytic base may be oriented and stabilized by a hydrogen
bonding network between Nδ of H303 with D331 and E360 (LtOMT), or D331 and D360 (HsOMT),
respectively33 (Fig. 3 and SI Appendix, Fig. S4).
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Figure 3. Structure modeling of LtOMT and HsOMT. Cartoon representations of
homology models and predicted active site cavities of LtOMT (A, C) and HsOMT (B, D) with
docked substrates DLD (carbon atoms in orange), DHZ (carbons in magenta) and co-product SAH
(carbons in green), all represented as sticks. The predicted active site H303 and the residues
stabilizing the active site (D331 and E360 for LtOMT, E331 and E360 for HsOMT) are also shown
as sticks (carbons in yellow). Residues in the active site cavity that were found to influence
substrate orientation are also shown as sticks (carbons in salmon for LtOMT, and grey for HsOMT),
and housed in the protein segments (orange on the cartoon diagrams of A and B) that were
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exchanged in hybrid enzymes H1 and H2 (Fig. 4). Key hydrogen bonding networks are displayed
as yellow dashes.
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Figure 4. Engineering the O-methylation regioselectivity of LtOMT. Left, schematic
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representation of chimeric and mutant enzymes created from LtOMT (in blue) and HsOMT (in
green), with the positions of amino acid replacements or fragment boundaries indicated. Middle,
product profiles (reversed-phase LC-MS traces recorded at 210 nm with a photodiode array detector)
of representative in vivo bioconversion reactions (DLD 1 as the substrate fed to cultures of S.
cerevisiae BJ5464-NpgA expressing the gene encoding the indicated enzyme). The identities of
the products were validated by comparison with isolated and structurally characterized compounds.
Right, total conversion (in percent) of DLD 1 into all corresponding methylated product congeners
are shown as bar graphs. Percentage distribution of the O-methylated products are represented as
pie charts (yields of the individual O-methylated products divided by the total yield of all products,
multiplied by 100). Color code: lasiodiplon 1a (blue), 5-O-methyl DLD 1b (green), and dimethoxy
DLD congener 1c (red). See SI Appendices Table S4 for percent conversion values (mean ± SD
from three independent experiments of three replicates each, n=9).
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Remodeling of the active site cavity alters regioselectivity.
To identify the residues that may influence the choice of the O-methylation target site in the
substrates, we built hybrid enzymes from LtOMT and HsOMT by fusion PCR, expressed these in
the S. cerevisiae chassis, and quantified the regioselectivity and the extent of O-methylation after
feeding DLD 1 as a model substrate. First, we created chimeras from LtOMT and HsOMT. In
chimera H1, amino acids 1-329 of LtOMT were replaced with those of HsOMT. Despite the
extensive replacement, the regioselectivity did not change, but the efficiency of H1 declined
dramatically (Fig. 4). The complementary hybrid H2 (amino acids 330-398 from HsOMT) retained
reasonable activity (26.1 ± 3.0% total conversion), but the regioselectivity completely switched to
that of HsOMT (i.e. para-OMT). Chimera H3 with a shorter HsOMT-derived segment (amino
acids 366-398) produced 1a:1b in a 3:1 ratio at 34.5 ± 0.8% conversion. These chimeras indicated
that regioselectivity is determined by the C-terminal end of the enzyme, and suggested that
extensive replacements are detrimental to enzymatic activity.
Next, we turned to structure-guided site-directed amino acid replacements to examine the
influence of residues located within 6Å of the substrates in the active site pockets of LtOMT and
HsOMT. Replacement of a cluster of four of these amino acids at the C-terminal end of LtOMT
with those of HsOMT (Q384K, G386R, W387H, and Q388H) by site-directed mutagenesis
provided mutant enzyme M1 that retained the overall high conversion rate of LtOMT (90.0 ± 2.9%
of substrate converted to methylated products). M1 preserved most of the ortho-OMT activity of
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LtOMT (lasiodiplodin 1a, 82.9 ± 0.8% of methylated products), but also gained HsOMT-type para-
OMT activity as shown by the accumulation of products 5-O-methyl DLD 1b (3.7 ± 0.8%) and
dimethoxy DLD congener 1c (13.4 ± 1.5%). Thus, replacement of these four amino acids is
sufficient to accommodate the model substrate within the active site cavity in orientations that
expose not only OH-3, but also OH-5 to the active site. Dissecting the influences of these amino
acids by creating all possible 3-point, 2-point and 1-point replacements (Fig. 4 and SI Appendix,
Table S4) indicated that W387H has the highest influence towards enabling a para-OMT activity.
Q388H (and to a lesser extent Q384K) play a supplementary role when combined with W387H,
while the effect of G386R is context-dependent. Thus, enzyme M4 with only the W387H mutation
shows para-OMT activity in addition to a robust ortho-OMT activity as evidenced by the
production of 7.7 ± 0.3% of dimethoxy DLD congener 1c at 76.6 ± 20.2% overall substrate
conversion. Addition of Q388H (as in M3) and further introduction of Q384K (as in M2) gradually
improves the para-OMT activity while preserving a strong ortho-OMT activity (1a:1b:1c = 4:1:1)
and continues to support high overall conversion (Fig. 4).
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Introduction of segment 330-366 of HsOMT into the four-point mutant M1 yielded enzyme
HM1 that acted exclusively as a para-OMT, indicating that this short region is decisive in setting
regioselectivity. Surprisingly, the chimeric mutant HM1 converted more of the DLD substrate to
5-O-methyl DLD 1b than HsOMT, the native para-OMT parent (58.3 ± 11.4% vs. 41.6 ± 1.2%).
We then investigated two residues of this segment that are situated within 6Å of the substrates.
Adding the F330V mutation to those already present in enzyme M1 (as in M5) improved the para-
OMT activity as evidenced by an increase of the 1b yield to 13.3 ± 5.5% (cf. 3.7 ± 0.8% for M1),
while the production of the 3,5-di-O-methoxy product 1c and the total conversion rate remained
unchanged. Introduction of the T356M mutation into M1 (as in M6) led to a dramatic reversal of
the regioselectivity with 5-O-methyl DLD 1b representing 74.3 ± 4.2% of the products and
lasiodiplodin 1a reduced to 10.0 ± 1.9%, albeit at the expense of a slight penalty in product yield
(70.1 ± 5.8% total conversion in M6 vs. 90.0 ± 2.9% in M1). Introduction of both changes into M1
afforded enzyme M7 (a six-point mutant) with an almost perfect para-OMT specificity (94.1 ± 1.0%
1b) and high overall conversion (93.7 ± 1.1%). Thus, mutant enzyme M7 far surpassed the parent
HsOMT as a DLD para-OMT. Surprisingly, mutant M8 with only a single amino acid replacement
(T356M) had an apparently balanced ortho-OMT/para-OMT activity with the three product
congeners present in roughly 1:1:1 at 80.1 ± 10.2% total conversion. Thus, this residue seems to
relax regioselectivity, and allows the substrate to assume either of the two binding poses with equal
likelihood. Introduction of a second mutation into enzyme M8 (W387H, cf. mutant M4) yielded
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M9 that afforded the 3,5-di-O-methyl-DLD 1c almost exclusively, and with a very high
productivity (96.4 ± 0.7% conversion of the substrate).
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To further characterize the native and the engineered enzymes, LtOMT, HsOMT and selected
variants of LtOMT (M1, M6-M9) were heterologously expressed in E. coli as His-tagged proteins
and purified to substantial homogeneity (SI Appendix, Fig. S5). The purified enzymes afforded
optimal conversion of the model substrate DLD 1 in the presence of 10 mM MgCl2. While NaCl
(0-100 mM), CaCl2 (10 mM) or MnCl2 (10 mM) did not influence the activity, EDTA, CuCl2 or
ZnSO4 (each at 10 mM) were strongly inhibitory. The enzymes showed a broad optimum at pH
7.5-9.5 in Tris or phosphate buffers, with the activity disappearing at pH 5.0 and above 10.0. The
temperature optimum was 30 °C, with residual activity still measurable at 4 °C and at 65 °C.
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The kinetic parameters for LtOMT and its variants with DLD 1 as the substrate are shown in
Table 1. HsOMT was found to be kinetically slow and the reaction did not reach saturation under
the conditions tested, thus Km and Vmax were not determined for this enzyme. Interestingly, the Km
of LtOMT and its five variants were similar. We speculate that the substrate binding cavity of
LtOMT is suitably spacious to accommodate the altered amino acid residues without reducing the
affinity of the mutant enzymes towards binding the model substrate. In contrast, the turnover rate
(kcat) and thus the catalytic efficiency (kcat/Km) of the LtOMT parent enzyme were more than two
orders of magnitude higher than those of the mutants. This may indicate that the altered substrate
poses adopted by the bound substrate decrease the rates of the formation of the transition state with
the mutant enzymes. Nevertheless, the turnover rates of the mutant enzymes were still similar to
those of many wild type secondary metabolite methyltransferases, such as the GenL gentamicin
C1/C2 methyltransferase (kcat 0.043 sec-1)34 or many flavone OMTs from plants (kcat often in the
range of 0.06 to 0.09 sec-1)15,16
.
Taken together, structure-based site-specific replacements of a few key “plasticity residues”
in the active site of LtOMT remodeled this regiospecific ortho-OMT enzyme to afford an almost
exclusive DLD para-OMT (M7, 6 amino acids replaced); an apparently unselective DLD ortho-
OMT/para-OMT (M8, single amino acid replacement); and a DLD o,p-di-OMT (M9, two amino
acids replaced). While homology modeling-based “keyhole surgery” was successful in modulating
the regioselectivity of LtOMT, protein crystallographic studies would be necessary to disentangle
the exact contributions of the individual amino acid replacements in forcing the substrates to adopt
altered binding poses, leading to modulated regioselectivities and catalytic efficiencies. Increasing
kinetic efficiency would also require further optimization. This is analogous to natural evolution
where functional innovation in the product and substrate spectrum is often the consequence of a
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few key amino acid changes, followed by the accumulation of compensatory mutations that
optimize the novel activity.35
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Table 1. Kinetic parameters of LtOMT and its engineered variants with DLD (1) as the
substrate
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Km
[mM]
Vmax
kcat
kcat/Km
[nmol sec-1 mg-1]
[sec-1]
[sec−1 mM−1]
LtOMT 1.58 ± 0.01
329.8 ± 102.8
1.5 ± 0.5
0.6 ± 0.1
1.0 ± 0.2
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1.4 ± 0.5
14.585 ± 4.547
0.068 ± 0.023
0.028 ± 0.002
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0.064 ± 0.022
9.209
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0.038
0.042
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1.15 ± 0.02
2.49 ± 0.22
1.40 ± 0.03
1.52 ± 0.30
kcat/Km was calculated using the averages of kcat and Km. Values represent the mean ± SD from
three independent experiments with three replicates each (n=9).
Regioselectivity of the engineered enzymes is influenced by the substrate.
The regiospecificities of the wild type LtOMT and HsOMT enzymes were found to be
independent of the substrates used (Fig. 2): for any given substrate accepted by the enzyme, only a
single O-methylated derivative was obtained, and the targeted phenolic OH was always ortho
(LtOMT) or para (HsOMT) to the aromatic carbon bearing the carbonyl “handle”. In contrast, the
mutant LtOMT enzymes possess both ortho-OMT and para-OMT activities to various degrees. To
discover whether the ratio of these activities is an intrinsic characteristic of these enzymes or if
regioselectivity is also influenced by the substrates, we selected five mutants (M1, M6-M9) with
characteristically different regioselective outcomes when acting on the DLD model substrate. We
conducted biocatalytic reactions with these enzymes using our panel of eight structurally varied
BDL substrates and their o-methoxy or p-methoxy congeners (Fig. 5, SI Appendix, Table S5).
The LtOMT variant enzymes processed DLD 1 and RDN 2, the two RAL12 scaffolds of the
panel in a similar manner. The T356M mutation in LtOMT variant M8 created a previously
nonexistent para-OMT activity against both the unmethylated compounds DLD 1 and RDN 2 and
their o-methoxy congeners lasiodiplodin 1a and 3-O-methyl RDN 2a. The ortho-OMT activity
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against the unmethylated and the p-methoxy congeners remained operational, thus the o,p-
dimethoxy congeners were produced through both the XXaXc and the XXbXc routes
(X=1 or 2). Adding the W387H mutation (as in M9) caused these o,p-dimethoxy congeners to
become the main products with substrates DLD 1 and RDN 2. This was the result of improvements
in both the para-OMT activity towards lasiodiplodin 1a and 3-O-methyl RDN 2a, and an increase
in the ortho-OMT activity towards 5-O-methyl DLD 1b and 5-O-methyl RDN 2b. Supplementing
the T356M and the W387H mutations with the Q384K, G386R, and Q388H changes (as in M6)
reduced activity towards the mono-methoxy congeners, but improved the para-OMT activity
towards the unmethylated substrates DLD 1 and RDN 2. As a result, the amounts of the dimethoxy
congeners were reduced, but the p-methoxy congeners 1b and 2b accumulated. Removing the
T356M mutation (as in M1) did not affect the ortho-OMT activity, but eliminated the para-OMT
activity against lasiodiplodin 1a and 3-O-methyl RDN 2a and limited it against DLD 1 and RDN
2. Introducing the F330V mutation into M6 (as in M7) did not seem to affect the ortho-OMT
activity against 1/1b and 2/2b. However, it improved the para-OMT activity against DLD 1 and
RDN 2 but not against the o-methoxy congeners 1a and 2a. As a result, 5-O-methyl DLD 1b
became the main product for the DLD scaffold, while 5-O-methyl RDN 2b production reached its
highest level for the RDN scaffold with this enzyme variant.
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The unmethylated RAL14 congeners were found to be less amenable than the RAL12
compounds towards para-O-methylation, perhaps as a consequence of the increased steric bulk of
the larger macrocycles. In particular, LCL 5 (and its o-methoxy congener 5a) remained completely
impervious to para-O-methylation just as with the other parent enzyme HsOMT, indicating that
the suggested inhibitory role of the enol tautomer of the C-9 carbonyl remains a problem even in
the context of an LtOMT scaffold. Thus, molding the active site cavity modulated only the ortho-
OMT activity against LCL 5. For the DHZ 3 and ZEA 4 scaffolds, no mutants were able to
accommodate the o-methoxy congeners 3a and 4a, perhaps because the increased bulk of the 3-
methoxy group obstructs access to the active site in the conformation that is conducive to the para-
OMT activity. Consequently, the XXaXc route (X=3 or 4) towards the dimethoxy analogs
remained unavailable for all enzyme variants. In contrast, the p-methoxy congeners 3b and 4b
remained excellent substrates for all LtOMT mutants, indicating that the methoxy group in the
ortho-OMT binding pose does not encounter similar steric restrictions. Consequently, the first-
stage products 5-O-methyl DHZ 3b and 5-O-methyl ZEA 4b did not accumulate but were
converted to the dimethoxy congeners through the XXbXc route (X=3 or 4).
The only DAL scaffold recognized as a substrate by the two parental enzymes was the 14-
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membered BDL, LLN 6, although the conversion rate was only moderate for both enzymes (37.7
± 1.4% for LtOMT and 30.7 ± 3.1% for HsOMT, Fig. 2). The introduced mutations allowed the
production of 7-O-methyl LLN 6a, 5-O-methyl LLN 6b, and the dimethoxy LLN congener 6c by
all engineered enzymes, with the mono-methoxy congeners 6a and 6b accepted as substrates for a
second round of O-methylation by all mutants except M1. However, overall conversion declined
precipitously to 2-5% with M1 and M6-M8, recovering to 15.8 ± 1.4% only with M9.
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Just as with the RAL14 scaffolds, the p-methoxy congeners of the two ARA (5-O-methyl
ARA7, 7b and 5-O-methyl ARA8, 8b) could be methylated by LtOMT and all its mutants, while
the o-methoxy congeners (3-O-methyl ARA7, 7a and 3-O-methyl ARA8, 8a) remained recalcitrant
towards subsequent methylation. Presumably, the bulky o-methoxy group prohibits the substrate
from binding in the active site cavity in a pose that could offer the para phenolic hydroxyl to the
active site base H303, but can be accommodated in the pose that leads to ortho-O-methylation.
Taken these together, it is striking that the apparent ortho-OMT/para-OMT preference of a
given enzyme variant is not an intrinsic and immutable property of the enzyme but emerges from
an intimate interplay with the various substrate scaffolds. Similar phenomena were also noted for
bacterial and mammalian catechol OMTs11–13. Product distribution among o-methoxy, p-methoxy
and o,p-dimethoxy derivatives is determined by two major factors: 1, how well the active site
accommodates the unmethylated substrate in the two poses that expose one or the other phenolic
hydroxyl to the active site; and 2, whether the o-methoxy and the p-methoxy first-stage reaction
products can be re-accommodated in the complementary pose that offers the free OH to the active
site. It is thus perhaps not surprising that mutant enzymes selected by using a single model substrate
(in this case, DLD 1) would reveal very different product profiles when offered a different substrate.
Analogous phenomena are often encountered in directed enzyme evolution projects, prompting the
formulation of the “first law of directed evolution”: you get what you screen for.36
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Figure 5. Different substrate scaffolds modulate the apparent regiospecificities of
selected engineered OMTs. Stacked polar bar charts show the product distributions among o-
methoxy (blue), p-methoxy (green) and o,p-dimethoxy (red) BDL congeners during biocatalytic
conversion of the indicated substrates (bold numbers above the bars) with S. cerevisiase BJ5464-
NpgA expressing LtOMT and its variants M1 and M6-M9. Percent values below the compound
numbers on these charts show the overall conversion percentage of the indicated substrate into all
corresponding methylated products. See SI Appendix, Table S5 for tabulated percent conversion
values as the mean ± SD from three independent experiments of three replicates each (n=9).
Scheme: Unmethylated substrates (X) may be converted to the o-methoxy congener (Xa) by the
ortho-OMT activity, and to the p-methoxy congener (Xb) by the para-OMT activity of the enzymes.
Either or both of these first-stage products may also be converted to the o,p-dimethoxy congener
(Xc) by the enzyme (X=1-8).
Conclusions
O-Methylation of reactive phenolic hydroxyl groups by regiospecific OMT enzymes is used
by BDL-producing fungi in nature to modulate the biological activities of these ecologically
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important small molecule natural products. For combinatorial biosynthesis of drug-like BDL
scaffolds in the laboratory, appropriate tailoring enzymes need to be employed to process unnatural
congeners produced by engineered polyketide biosynthetic pathways. Amongst these tailoring
enzymes, OMTs need to display significant promiscuity to accept structurally varied BDL scaffolds
as substrates, while these enzymes must also offer appropriate regioselectivity to deliver the desired
methoxy regioisomer. Our work shows that two orthologous OMT enzymes from different BDL
biosynthetic pathways possess considerable substrate flexibility but display strict orthogonal
regiospecificity. Protein homology modeling and substrate docking reveals that nature used subtle
alterations of the amino acids lining the active site cavity to force the substrates to adopt different
binding poses. In turn, this exposes distinct phenolic hydroxyls to the conserved catalytic base,
accounting for the observed orthogonal regiospecificity. This apparent plasticity of the active site
cavity architecture then allows us to re-program the regioselectivity of BDL OMT enzymes by
adjusting just a few amino acids in a keyhole surgery-like manner. Doing so, we can transpose the
strict regiospecificity of a native OMT to generate a mutant enzyme that produces the orthogonal
p-methoxy derivative of the model substrate almost exclusively. We can also engineer a mutant
enzyme that apparently eschews regioselectivity towards the model substrate to afford o-methoxy,
p-methoxy, and o,p-dimethoxy congeners in equal proportions; or another one that specializes in
recursive methylation to yield mostly the o,p-dimethoxy derivative. We also show that apparently
similar product distributions may result from different scenarios involving dissimilar recognition
and processing of the unmethylated, the o-methoxy, and the p-methoxy substrates by the ortho-
OMT and the para-OMT activities engineered into the same enzyme chassis. As their active site
cavity is being remodeled, the enzymes may acquire or lose one or the other activity, and come to
accept or reject certain substrates. In accord with these dynamic changes, the regiospecificities and
the resulting product ratios of the mutant OMT enzymes turn out to be context dependent, as they
are modulated by the complex interactions of the structurally varied substrates with the enzymes.
Thus, engineering an OMT to generate a desired methoxy regioisomer of a given “unnatural
product” during combinatorial biosynthesis is analogous to enzyme evolution in nature where
catalytic innovations emerge in the context of a particular molecule that provides fitness advantage
to the producer, and is accomplished by a few key mutations in “plasticity residues” followed by
the acquisition of supplementary mutations to optimize the novel activity. Recapturing this process
in synthetic biology by directed enzyme evolution towards improved catalytic efficiency, coupled
with chassis metabolic engineering and fermentation process optimization would doubtless be also
necessary to deploy engineered OMTs for practical, industrial scale applications, as was seen with
the recent development of a commercial process for vanillin production that also employs an
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OMT.40–42 Even so, extension of the scalable, sustainable and tunable enzymatic approach
described here towards the regioselective O-methylation of a wider variety of phenolic polyketide
natural products, such as the isocoumarins, isochromanones anthraquinoids, and the
flavonoids7,10,43,44 offers advantages over the well-known chemical methods that require high
temperature/pressure and longer reaction times often leading to undesired C-methylated
byproducts.8,9
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Data availability
Experimental and bioinformatic procedures, descriptions on the isolation and structure
elucidation of compounds, spectroscopic data, tabulated biocatalytic conversion rates,
supplementary figures including additional structures, dissection of demethylation routes, and
protein topology and docking models are all available in the Supplementary Information. All other
data are available from the authors upon reasonable request.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (31570093
and 31870076 to Y. X and 21807110 to C. W.), the National Program of China for Transgenic
Research (No. 2016ZX08009003-002 to M. L.), the National Basic Research Program of China
(2015CB755700 to Y. X. and M. L.), China Postdoctoral Science Foundation (2018M640208 to C.
W.), the Joint Genomics Institute of the U.S. Department of Energy (WIP ID 1349 to I. M.), and
the National Institutes of Health (NIGMS 5R01GM114418 to I. M.). We thank the Beijing Center
for Physical and Chemical Analysis for providing access to their NMR device. I. M. has disclosed
financial interests in TEVA Pharmaceuticals Hungary and DSM Nutritional Products, LLC, which
are unrelated to the subject of the research presented here. All other authors declare no competing
financial interests.
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References
5
6
7
8
9
(1)
(2)
Wessjohann, L. A.; Keim, J.; Weigel, B.; Dippe, M. Alkylating enzymes. Curr. Opin. Chem. Biol.
2013, 17, 229–235.
Koirala, N.; Thuan, N. H.; Ghimire, G. P.; Thang, D. V.; Sohng, J. K. Methylation of flavonoids:
Chemical structures, bioactivities, progress and perspectives for biotechnological production. Enzyme
Microb. Technol. 2016, 86,103–116.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(3)
(4)
Liscombe, D. K.; Louie, G. V.; Noel, J. P. Architectures, mechanisms and molecular evolution of
natural product methyltransferases. Nat. Prod. Rep. 2012, 29,1238–1250.
Wessjohann L.A.; Dippe M.; Tengg M.; Gruber-Khadjawi M. Methyltransferases in biocatalysis.
Cascade biocatalysis: integrating stereoselective and environmentally friendly reactions, 1st ed.;
Wiley: Somerset, NJ, 2014; pp 393–425.
(5)
Henry, K. M.; Townsend, C. A. Ordering the reductive and cytochrome P450 oxidative steps in
demethylsterigmatocystin formation yields general insights into the biosynthesis of aflatoxin and
related fungal metabolites. J. Am. Chem. Soc. 2005, 127, 3724–3733.
(6)
(7)
Du, Y. L.; Ding T.; Ryan, K. S. Biosynthetic O-methylation protects cladoniamides from self-
destruction. Org. Lett. 2013, 15, 2538–2541.
Struck, A. W.; Thompson, M. L.; Wong, L. S.; Micklefield, J. S-Adenosyl-methionine-dependent
methyltransferases: Highly versatile enzymes in biocatalysis, biosynthesis and other biotechnological
applications. ChemBioChem. 2012, 13, 2642–2655.
(8)
(9)
Shen, Z. L.; Jiang, X. Z.; Mo, W. M.; Hu, B. X.; Sun, N. Catalytic O-methylation of phenols with
dimethyl carbonate to aryl methyl ethers using BMIm Cl. Green Chem. 2005, 7, 97–99.
Basak, A.; Nayak, M. K.; Chakraborti, A. K. Chemoselective O-methylation of phenols under non-
aqueous condition. Tetrahedron Lett. 1998, 39, 4883–4886.
(10) Bennett, M. R.; Shepherd, S. A.; Cronin, V. A.; Micklefield, J. Recent advances in methyltransferase
biocatalysis. Curr. Opin. Chem. Biol. 2017, 37, 97–106.
(11) Wang, J. H.; Pichersky, E. Identification of specific residues involved in substrate discrimination in
two plant O-methyltransferases. Arch. Biochem. Biophys. 1999, 368, 172–180.
(12) Schmidt, A.; Li, C.; Shi, F.; Jones, A. D.; Pichersky, E. Polymethylated myricetin in trichomes of the
wild tomato species Solanum habrochaites and characterization of trichome-specific 3'/5'- and 7/4'-
myricetin O-methyltransferases. Plant Physiol. 2011, 155, 1999–2009.
(13) Lee, A.; Park, E. B.; Lee, J.; Choi, B. S.; Kang, S. J. The N terminus of cGAS de-oligomerizes the
cGAS: DNA complex and lifts the DNA size restriction of core-cGAS activity. FEBS letters. 2017,
591, 954–961.
(14) Law, B. J.; Bennett, M. R.; Thompson, M. L.; Levy, C.; Shepherd, S. A.; Leys, D.; Micklefield, J.
Effects of active-site modification and quaternary structure on the regioselectivity of catechol-O-
methyltransferase. Angew. Chem., Int. Ed. 2016, 55, 2683–2687.
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Page 24 of 32
1
2
3
4
5
6
7
8
(15) Berim, A.; Hyatt, D. C.; Gang, D. R. A set of regioselective O-methyltransferases gives rise to the
complex pattern of methoxylated flavones in sweet basil. Plant Physiol. 2012, 160, 1052–1069.
(16) Joe, E. J.; Kim, B. G.; An, B. C.; Chong, Y.; Ahn, J. H. Engineering of flavonoid O-methyltransferase
for a novel regioselectivity. Mol. Cells. 2010, 30, 137–141.
(17) Wils, C. R.; Brandt, W.; Manke, K.; Vogt, T. A single amino acid determines position specificity of
an Arabidopsis thaliana CCoAOMT-like O-methyltransferase. FEBS letters. 2013, 587, 683–689.
(18) Shen, W.; Mao, H.; Huang, Q.; Dong, J. Benzenediol lactones: A class of fungal metabolites with
diverse structural features and biological activities. Eur. J. Med. Chem. 2015, 97, 747–777.
(19) Patocka, J.; Soukup, O.; Kuca, K. Resorcylic acid lactones as the protein kinase inhibitors, naturally
occuring toxins. Mini Rev. Med. Chem. 2013, 13, 1873–1878.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(20) Liu, Z.; Zheng, X.; Wang, Y.; Tang, M.; Chen, S.; Zhang, F.; Li, L.; Zhang, C.; Sun, Y. Lignans and
isoflavonoids from the stems of Pisonia umbellifera. RSC Adv. 2018, 8, 16383–16391.
(21) Xu, Y.; Zhou, T.; Espinosa-Artiles, P.; Tang, Y.; Zhan, J.; Molnár, I. Insights into the biosynthesis of
12-membered resorcylic acid lactones from heterologous production in Saccharomyces cerevisiae.
ACS Chem. Biol. 2014, 9, 1119–1127.
(22) Xu, Y.; Espinosa-Artiles, P.; Schubert, V.; Xu, Y.-M.; Zhang, W.; Lin, M.; Gunatilaka, A. L.;
Süssmuth, R.; Molnár, I. Characterization of the biosynthetic genes for 10,11-dehydrocurvularin, a
heat shock response-modulating anticancer fungal polyketide from Aspergillus terreus. Appl. Environ.
Microbiol. 2013, 79, 2038–2047.
(23) Wang, S.; Xu, Y.; Maine, E. A.; Wijeratne, E. K.; Espinosa-Artiles, P.; Gunatilaka, A. L.; Molnár, I.
Functional characterization of the biosynthesis of radicicol, an Hsp90 inhibitor resorcylic acid lactone
from Chaetomium chiversii. Chem. Biol. 2008, 15, 1328–1338.
(24) Xu, Y.; Zhou, T.; Zhang, S.; Xuan, L. J.; Zhan, J.; Molnár, I. Thioesterase domains of fungal
nonreducing polyketide synthases act as decision gates during combinatorial biosynthesis. J. Am.
Chem. Soc. 2013, 135, 10783–10791.
(25) Bai, J.; Lu, Y.; Xu, Y.-M.; Zhang, W.; Chen, M.; Lin, M.; Gunatilaka, A. L.; Xu, Y.; Molnár, I.
Diversity-oriented combinatorial biosynthesis of hybrid polyketide scaffolds from azaphilone and
benzenediol lactone biosynthons. Org. Lett. 2016, 18, 1262–1265.
(26) Xu, Y.; Zhou, T.; Zhang, S.; Espinosa-Artiles, P.; Wang, L.; Zhang, W.; Lin, M.; Gunatilaka, A. L.;
Zhan, J.; Molnár, I. Diversity-oriented combinatorial biosynthesis of benzenediol lactone scaffolds by
subunit shuffling of fungal polyketide synthases. Proc. Natl. Acad. Sci. U.S.A. 2014, 111, 12354–
12359.
(27) Xu, Y.; Zhou, T.; Zhou, Z.; Su, S.; Roberts, S. A.; Montfort, W. R.; Zeng, J.; Chen, M.; Zhang, W.;
Lin, M.; Zhan, J.; Molnár, I. Rational reprogramming of fungal polyketide first-ring cyclization. Proc.
Natl. Acad. Sci. U.S.A. 2013, 110, 5398–5403.
(28) Xie, L.; Zhang, L.; Wang, C.; Wang, X.; Xu, Y.-M.; Yu, H.; Wu, P.; Li, S.; Han, L.; Gunatilaka, A.
L.; Wei, X.; Lin, M.; Molnár, I.; Xu, Y. Methylglucosylation of aromatic amino and phenolic moieties
ACS Paragon Plus Environment

Page 25 of 32
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1
2
3
4
5
of drug-like biosynthons by combinatorial biosynthesis. Proc. Natl. Acad. Sci. U.S.A. 2018, 115,
E4980–E4989.
6
7
8
9
(29) Reeves, C. D.; Hu, Z.; Reid, R.; Kealey, J. T. Genes for biosynthesis of the fungal polyketides
hypothemycin from Hypomyces subiculosus and radicicol from Pochonia chlamydosporia. Appl.
Environ. Microbiol. 2008, 74, 5121–5129.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(30) Dai, Q.; Jiang, Y.; Yu, J. T.; Cheng, J. Peroxide: A novel methylating reagent. Synthesis. 2016, 48,
329–339.
(31) Singh, S.; Chang, A.; Goff, R. D.; Bingman, C. A.; Grüschow, S.; Sherman, D. H.; Phillips, G. N.,
Jr.; Thorson, J. S. Structural characterization of the mitomycin 7-O-methyltransferase. Proteins. 2011,
79, 2181–2188.
(32) Chang, A.; Singh, S.; Bingman, C. A.; Thorson, J. S.; Phillips, G. N., Jr. Acta. Structural
characterization of CalO1: a putative orsellinic acid methyltransferase in the calicheamicin-
biosynthetic pathway. Crystallogr. D. 2011, 67, 197–203.
(33) Zubieta, C.; He, X. Z.; Dixon, R. A.; Noel, J. P. Structures of two natural product methyltransferases
reveal the basis for substrate specificity in plant O-methyltransferases. Nat. Struct. Biol. 2001, 8, 271–
279.
(34) Li, S.; Guo, J.; Reva, A.; Huang, F.; Xiong, B.; Liu, Y.; Deng, Z.; Leadlay, P. F.; Sun, Y.
Methyltransferases of gentamicin biosynthesis. Proc. Natl. Acad. Sci. U.S.A. 2018, 115, 1340–1345.
(35) Newton, M. S.; Arcus, V. L.; Gerth, M. L.; Patrick, W. M. Enzyme evolution: innovation is easy,
optimization is complicated. Curr. Opin. Chem. Biol. 2018, 48, 110–116.
(36) Hibbert, E. G.; Dalby, P. A. Directed evolution strategies for improved enzymatic performance.
Microb. Cell Fact. 2005, 4, 29.
(37) Ma, S. M.; Li, J. W.-H.; Choi, J. W.; Zhou, H.; Lee, K. M.; Moorthie, V. A.; Xie, X.; Kealey, J. T.;
Silva, N. A. D.; Vederas, J. C.; Tang, Y. Complete reconstitution of a highly reducing iterative
polyketide synthase. Science. 2009, 326, 589–592.
(38) Biasini, M.; Bienert, S.; Waterhouse, A.; Arnold, K.; Studer, G.; Schmidt, T.; Kiefer, F.; Cassarino,
T. G.; Bertoni, M.; Bordoli, L.; Schwede, T. SWISS-MODEL: modelling protein tertiary and
quaternary structure using evolutionary information. Nucleic. Acids Res. 2014, 42, W252–258.
(39) Holm, L.; Laakso, L. M. Dali server update. Nucleic. Acids Res. 2016, 44, W351–355.
(40) Hansen, E. H.; Moller, B. L.; Kock, G. R.; Bunner, C. M.; Kristensen, C.; Jensen, O. R.; Okkels, F.
T.; Olsen, C. E.; Motawia, M. S.; Hansen, J. De novo biosynthesis of vanillin in fission yeast
(Schizosaccharomyces pombe) and baker's yeast (Saccharomyces cerevisiae). Appl. Environ.
Microbiol. 2009, 75, 2765–2774.
(41) Brochado, A. R.; Matos, C.; Moller, B. L.; Hansen, J.; Mortensen, U. H.; Patil, K. R. Improved vanillin
production in baker's yeast through in silico design. Microb. Cell Fact. 2010, 9, 84.
(42) Hayden, E. C. Synthetic-biology firms shift focus. Nature. 2014, 505, 598.
(43) Chandler, I. M.; McIntyre, C. R.; Simpson, T. J. Structural revision and synthesis of LL-D253α and
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related chromanone fungal metabolites. J. Chem. Soc., Perkin Trans. 1. 1992, 2271–2284.
(44) Chen, Z.-G.; Fujii, I.; Ebizuka, Y.; Sankawa, U. Emodin O-methyltransferase from Aspergillus
terreus. Arch. Microbiol. 1992, 158, 29–34.
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Table of Contents Graphic
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Figure 1. Total biosynthesis of O-methylated BDLs using LtOMT and HsOMT.
84x44mm (300 x 300 DPI)
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Figure 2. Combinatorial tailoring with LtOMT and HsOMT.
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Figure 3. Structure modeling of LtOMT and HsOMT.
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